Grant and Publication Support Guide

In order to facilitate the listing of NuGEN kits and reagents in grant applications, as well as scientific publications/abstracts, this guide provides the latest information about our products and technology for microarray, qPCR, and Next Generation Sequencing (NGS) sample preparation.

Please note, that as specified in the Application Guide for NIH grants, researchers are required to itemize consumable reagents that exceed $1,000 in direct costs for project budgets.

I. Description of NuGEN Products

The text below describing NuGEN products may be copied directly into grant applications or scientific submissions.

DNA Sample Preparation

Ovation® WGA System (Part No.: 6100)
The Ovation WGA System offers a robust and simple linear amplification technology based on SPIA (Single Primer Isothermal Amplification). This amplification system faithfully replicates genomic DNA, enabling a variety of downstream applications including copy number change analysis using real-time quantitative PCR (qPCR) or microarrays.

RNA Sample Preparation

WT-Ovation™ FFPE System V2 (Part No.: 3400)
The WT-Ovation FFPE System V2 is a whole transcriptome RNA amplification system based on Ribo-SPIA technology for gene expression analysis of limited and degraded FFPE RNA samples. Amplification is initiated both at the 3' end and randomly throughout the entire transcriptome, enabling downstream analysis by real-time quantitative PCR (qPCR) or microarrays.

WT-Ovation™ Pico System (Part No.: 3300)
A whole transcriptome RNA Amplification System based on Ribo-SPIA technology initiating both at the 3' end and randomly throughout the entire transcriptome. For use with limited or degraded RNA samples such as LCM, FACS, biopsies, FNA, and other limited biological specimens. Enables downstream analysis by real-time quantitative PCR (qPCR) or microarrays.

Ovation® RNA Amplification System V2 (Part No. : 3100)
This 3'-focused RNA amplification product based on Ribo-SPIA technology provides a simple automation-friendly approach for use with intact and non-compromised RNA samples. The add and incubate process is amenable to high throughput applications, and enables downstream analysis by real-time quantitative PCR (qPCR) or microarrays.

Ovation® Whole Blood Solution
Consisting of optimized products and protocols for global gene expression profiling of whole blood RNA without the need for globin reduction procedures. The fast, simple, automation friendly workflow based on Ribo-SPIA technology enables downstream analysis by real-time quantitative PCR (qPCR) or microarrays.

WT-Ovation™ RNA Amplification System (Part No.: 2210)
This whole transcriptome RNA Amplification product based on Ribo-SPIA technology is specifically designed for pre-amplification approaches prior to real-time quantitative PCR (qPCR) applications. Amplification is initiated both at the 3' end and randomly throughout the transcriptome providing flexibility in qPCR assay design.

WT-Ovation™ One-Direct System (Part No.: 3500)
The One-Direct System enables a whole-transcriptome view from one or a few cells with the option to go directly from cell lysate eliminating the necessity for inefficient and time-consuming RNA purification. The process based on Ribo-SPIA technology allows amplification and target preparation ready for qPCR or microarray analysis in a single day.

Ovation® RNA-Seq System (Part No.: 7100)
The Ovation RNA-Seq System is based on Ribo-SPIA technology, and provides a fast and simple method for preparing amplified cDNA from total RNA. The double-stranded cDNA product of the Ovation RNA-Seq System is optimized for the generation of libraries for Next Generation Sequencing platforms.

Applause™ WT-Amp ST and WT-Amp Plus ST Systems (Part Nos.: 5500 and 5510)
The Applause Systems offer a robust and simple linear amplification technology based on Ribo-SPIA technology with sample sizes starting at 50 ng total RNA. The product delivers a single-tube, single purification, add-and-incubate assay that can be completed in a single day.

Applause™ 3'-Amp System (Part No.: 5100)
The Applause 3'-Amp System offers a robust and simple linear amplification technology based on Ribo-SPIA technology with sample sizes starting at 50 ng total RNA. The product delivers a single-tube, single purification, add-and-incubate assay that can be completed in a single day.

Encore™ Biotin Module (Part No.: 4200)
Designed for fragmentation and biotin labeling of cDNA, this module is optimized for use with Affymetrix GeneChip® micorarrays. In a simple, robust and automatable add and incubate process, target preparation is completed without the need for any purification steps.

WT-Ovation™ Exon Module (Part No.: 2000)
This module uses whole transcriptome amplified cDNA input to create an ST-cDNA sample for expression analysis on sense-target Affymetrix GeneChip® Gene ST and Exon ST arrays. The method does not require ribosomal RNA removal and is readily automatable.

II. SPIA® and Ribo-SPIA® Technology

The text below summarizes the linear amplification technology used in each of the NuGEN Ovation, WT-Ovation, and Applause amplification products and may be copied directly into grant applications or scientific submissions.

SPIA Amplification Process
Single Primer Isothermal Amplification (SPIA) is a linear DNA amplification process that uses a DNA/RNA chimeric primer (SPIA Primer), DNA polymerase and RNase H in a homogeneous isothermal assay providing highly efficient amplification of DNA sequences. (1) Genomic DNA (gDNA) is denatured, and a chimeric DNA/RNA primer mix (WG Primer) hybridizes uniformly across the input gDNA. The RNA portion of the WG Primer includes a unique sequence that serves as the tag for the subsequent linear SPIA process. DNA polymerase creates the first SPIA template strand by extending from the chimeric WG Primer. The second strand is then synthesized in the same tube without additional reagent addition to form the resulting double-stranded DNA (SPIA Template) with a DNA/RNA heteroduplex at one end that contains the unique tag sequence; (2) The SPIA Template is purified away from the excess primers with magnetic beads; (3) In the SPIA reactions, RNase H is used to degrade the RNA portion of the DNA/RNA heteroduplex introduced during the SPIA Template Synthesis reaction. This process results in exposure of a DNA tag sequence that is now available for binding to a second SPIA Primer. DNA polymerase then initiates replication at the 3' end of the primer, displacing the existing forward strand. The RNA portion at the 5' end of the newly synthesized strand is again removed by RNase H, exposing part of the unique priming site for initiation of the next round of DNA synthesis. The process of SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage is repeated, resulting in rapid accumulation of DNA with sequence complementary to the original gDNA.

Ribo-SPIA 3' Amplification Process
Ribo-SPIA technology is a three-step process that generates micrograms of cDNA from nanograms of total RNA. (1) First Strand Synthesis: Single stranded cDNA is prepared from total RNA using a chimeric DNA/RNA primers and reverse transcriptase. The primer has a DNA portion that hybridizes specifically to poly(A) sequences. The primer has a 3' DNA portion that hybridizes to the mRNA and a unique 5' RNA portion that does not hybridize to the mRNA. The resulting cDNA/mRNA complex includes the unique RNA sequence at the 5' end of the cDNA; (2) Second Strand Synthesis: After partial fragmentation of mRNA in the cDNA/mRNA complex, DNA polymerase generates a second strand, including DNA complementary to the 5' RNA unique sequence incorporated into the first strand, resulting in a double-stranded cDNA with an RNA/DNA heteroduplex of unique sequence at one end; (3) SPIA Amplification: The isothermal linear amplification step uses an additional DNA/RNA chimeric primer, DNA polymerase and RNase H in an isothermal reaction. RNase H removes the unique RNA sequence in the double-stranded cDNA revealing a site for binding the DNA/RNA chimeric primer. DNA polymerase synthesizes cDNA starting at the 3' end of the primer and displacing the existing forward strand. RNA at the 5' end of the newly synthesized strand is again cleaved by RNase H, exposing the priming site for initiation of the next round of DNA synthesis. The entire process is repeated continuously with multiple DNA polymerase molecules participating in DNA synthesis along the same template molecule, leading to the rapid accumulation of micrograms of amplified single stranded (opposite sense) cDNA products.

Ribo-SPIA Whole Transcriptome (WT) Amplification Process
Ribo-SPIA technology is a three-step process that generates micrograms of cDNA from nanograms of total RNA. Amplification occurs across the whole transcript, resulting in successful amplification of mRNA species, even including highly degraded samples. (1) First Strand Synthesis: Single stranded cDNA is prepared from total RNA using a unique mix of chimeric DNA/RNA primers and reverse transcriptase. This mix contains both random as well as poly T primers to achieve comparable representation across the entire transcriptome. The primer has a 3' DNA portion that hybridizes to the mRNA and a unique 5' RNA portion that does not hybridize to the mRNA. The resulting cDNA/mRNA complex includes the unique RNA sequence at the 5' end of the cDNA; (2) Second Strand Synthesis: After partial fragmentation of mRNA in the cDNA/mRNA complex, DNA polymerase generates a second strand, including DNA complementary to the 5' RNA unique sequence incorporated into the first strand, resulting in a double-stranded cDNA with an RNA/DNA heteroduplex of unique sequence at one end; (3) SPIA Amplification: The isothermal linear amplification step uses an additional DNA/RNA chimeric primer, DNA polymerase and RNase H in an isothermal reaction. RNase H removes the unique RNA sequence in the double-stranded cDNA revealing a site for binding the DNA/RNA chimeric primer. DNA polymerase synthesizes cDNA starting at the 3' end of the primer and displacing the existing forward strand. RNA at the 5' end of the newly synthesized strand is again cleaved by RNase H, exposing the priming site for initiation of the next round of DNA synthesis. The entire process is repeated continuously with multiple DNA polymerase molecules participating in DNA synthesis along the same template molecule, leading to the rapid accumulation of micrograms of amplified single stranded (opposite sense) cDNA products.

III. Publications

A searchable database of scientific publications using NuGEN products:
Publications

IV. NuGEN Patents and Trademarks

The Ovation and Applause System family of products and methods is covered by U.S. Patent Nos. 6,692,918, 6,251,639, 6,946,251 and 7,354,717, and other issued and pending patents in the U.S. and other countries. NuGEN®, Ovation®, SPIA®, Ribo-SPIA®, WT-Ovation™, Encore™, FL-Ovation™, Applause™ and Imagine More from Less are trademarks or registered trademarks of NuGEN Technologies, Inc.

V. General Resources for Grant Writing and Research Funding

National Institutes of Health
NIH Grant Cycle:
http://www.niaid.nih.gov/ncn/grants/cycle/default.htm
NIH Funding Opportunities:
http://grants.nih.gov/grants/guide/index.html
NIH Grant Programs:
http://grants.nih.gov/grants/funding/funding_program.htm
NIH Forms and Applications:
http://grants.nih.gov/grants/forms.htm

National Cancer Institute
NCI Funding Opportunities:
http://deainfo.nci.nih.gov/funding.htm

National Institute for Allergy and Infectious Disease
NIAID Funding Opportunities:
http://www.niaid.nih.gov/ncn/newsletters/default.htm